Process for using endogenous enkephalins and endorphins to stimulate the immune system

ABSTRACT

A process of stimulating and promoting the natural immune system (T cells and NK cells) resisting and inhibiting the growth of tumorous cells as well as resisting infections (virus bacteria, and fungi) is disclosed. Endogenous endorphins are disclosed as the agent for stimulating the immune system.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of the parent application Ser. No. 308,287 filed the 5th day of October, 1981.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a process of stimulating and promoting the natural immune system (T cells and NK cells) resisting and inhibiting the growth of tumorous cells as well as resisting infections (virus, bacteria, and fungi). More specifically this invention relates to the in vivo use of leucine-enkephalin and methionine-enkephalin or the like.

2. Description of the Prior Art

Since the discovery, isolation and the complete analysis of the amino acid sequence of specific, endogenous, highly reactive and incredibly powerful peptides (e.g. interferon, endorphins and enkephalins), a rebirth and revitalization of interest and activity in biochemical research has taken place. During the last decade the industrial world's race to be the first to commercially synthesize interferon is not only a daily topic on Wall Street but is also well known to the layman and casual observer. Perhaps as at no other time in history has the phrase "the miracles of modern medicine" been more applicable as a geniuine expectation.

Consistent with the contemporary so-called `lock and key` theory, the existence of specific receptors in the brain for morphine-like substances has been established and corresponding endogenous specific ligands have been located and identified to be two pentapeptides, methionine-enkephalin and leucine-enkephalin (Hughes et al, Nature, 258 577-579 (1975); Simantov et al, Proc. Natl. Acad. Sci., U.S.A., 73 2515-2519 (1976); Kosterlitz, Opiates and Endogenous Opioid Peptides, Elsevier/North Holland Biomedical Press (1976)). Other larger peptides, the endorphins, have also been found to bind to morphine receptors (Li et al, Nature 260 622-24 (1976); Cox et al, Proc. Natl. Acad. Sci., U.S.A., 73 1821-23 (1976); Segal et al, Science 198 411-413 (1977)). Subsequent studies elaborated on the analgesic activity in various animal models by the intracerebral route of administration. However, there appeared to be a lack of analgesic activity when the peptides were administered by the intravenous (or intramuscular or intraperitoneal) routes of administration. This dichotomy of analgesic activity by different routes of administration (brain versus peripheral) was explained in terms of blood-brain barrier differences as well as a rapid rate of metabolism in plasma.

However, pronounced activity by systemic administration was discovered by Plotnikoff et al (Life Sci. 19 1283-1288 (1976)), who showed that the enkephalins-endorphins exhibited marked activity as tranquilizers and antidepressants. More important, Plotnikoff demonstrated that the enkephalins were extremely active in potentiating the central effects of dopamine.

In 1979, Wybran et al reported that normal human blood T lymphocytes bear surface receptor-like structures for methionine-enkephalin and recent discoveries further support the view that T-cell lymphocytes were covered with enkephalin and endorphin receptor sites.

In the same year, the New York Academy of Sciences Symposium on Subcellular Factors in Immunity (Volume 332, Dec. 28, 1979) disclosed that the thymus gland of animals and humans was secreting peptides (thymosin and others) that controlled activities of T-cell lymphocytes and associated cells. These thymus peptides were found to have anti-neoplastic effects in animals and humans. In addition, the thymus peptides were found to control the aging process. Finally, the thymus peptides appear to regulate `auto-immune responses` of lymphocyte particles.

SUMMARY OF THE INVENTION

In view of the above, I have discovered a process for stimulating the natural immune system resisting tumerous growth and infections (viruses, bacteria, and fungi) comprising the step of treating the tumorous growth and infections, in vivo, with an endogenous enkephalin. The invention further provides that the endogenous enkephalin be leucine-enkephalin or methionine-enkephalin and that they be administered at a dosage rate from about a quarter of a miligram of enkephalin per kilogram of body weight to as low as about 10⁻¹⁴ mg/kg.

It is an object of the present invention to provide a process for the use of naturally occurring peptides to promote and stimulate the immune system's resistance to neoplastic cellular growth and infection. It is a further object to provide a process for using, in vivo, leucine-enkephalin and methionine-enkephalin to stimulate the natural immune system's anti-neoplastic, anti-viral, anti-bacterial, anti-fungal and anti-parasitic activity. Fulfillment of these objects and the presence and fulfillment of other objects will be apparent upon complete reading of the specification and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the in vivo effect of methionine-enkephalin on the NK cell activity of human blood from normal volunteers.

FIG. 2 illustrates the in vivo effect of leucine-enkephalin on the NK cell activity of human blood from normal volunteers.

FIG. 3 illustrates the in vivo effect of methionine-enkephalin on the NK cell activity of human blood from cancer patients.

FIG. 4 illustrates the in vivo effect of leucine-enkephalin on the NK cell activity of human blood from cancer patients.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Starting with the two facts: (1) T-cell lymphocytes are covered with enkephalin and endorphin receptors and (2) the thymus gland of animals and humans secretes peptides (thymosin and others) that control the activities of T-cell lymphocytes and associated cells, I propose a working hypothesis that the hypothalamus secretes the releasing factor (corticotrophin releasing factor--CRF) to the pituitary gland (storehouse of beta-endorphin and ACTH). Therefore, the beta-endorphins' and ACTH's release are controlled by factors from the hypothalamus. The release of beta-endorphin and ACTH from the pituitary results in stimulation of the thymus and adrenals releasing enkephalins and endorphins and the entire immune cascade of responses. Beta-endorphin is probably also metabolized in the bloodstream into its numerous fragments (alpha- and gamma-endorphin as well as the smaller enkephalins). These smaller fragments are active in stimulating the immune systems.

In order to test the hypothesis and to establish the efficacy of these smaller fragments, the following series of in vivo experiments were designed and performed, using laboratory mice inoculated with tumorous cells and then treated with leucine-enkephalin and methionine-enkephalin. Groups of BDF₁ female mouse strain (16-20 gms) were inoculated with L1210 (leukemia) turmor cells (1×10⁴, 1×10³, or 1×10² cells) and observed for survival. The L1210 line of tumor cells was maintained in DBA/2 host mice. Ascitic fluid from the DBA/2 mice was used to inoculate the BDF₁ mice transferred to RPMI 1640 buffer at pH 8-9).

EXAMPLE I

Three separate experiments were conducted utilizing ten mice per group. The first experiment involved inoculating all mice with 1×10⁴ cells, the second with 1×10³ cells, and the third with 1×10² cells. Control groups were administered vehicle subcutaneously daily, while the methionine-enkephalin groups and the leucine-enkephalin groups were administered 10 mg/kg s.c. daily. The survival rate of the mice was monitored and the corresponding data for the three experiments are presented in TABLES I, II, and III respectively.

                  TABLE I                                                          ______________________________________                                         1 × 10.sup.4 L1210 Tumor Cells in                                        BDF.sub.1 Mice -                                                               Effects of Enkephalins                                                                 Methionine-                 Leucine-                                           Enkephalin                  Endkephalin                                Days    10 mg/kg s.c.*    Controls  10 mg/kg s.c.*                             ______________________________________                                         11      6      died       5   died  8    died                                  12      1      died       2   died  0    died                                  13      2      died       3   died  2    died                                           -9    died        --10                                                                              died   --10                                                                               died                                          10                10        10                                         ______________________________________                                          *Enkephalin injections started on day 2                                  

                  TABLE II                                                         ______________________________________                                         1 × 10.sup.3 L1210 Tumor Cells in                                        BDF.sub.1 Mice -                                                               Effects of Enkephalins                                                                 Methionine-                 Leucine-                                           Enkephalin                  Enkephalin                                 Days    10 mg/kg s.c.    Controls   10 mg/kg s.c.                              ______________________________________                                         13      0      died      2   died   2    died                                  14      4      died      2   died   4    died                                  15      3      died      5   died   0    died                                  16      1      died      1   died   3    died                                  17      1      died      0   died   0    died                                  18      1      died      0   died   1    died                                           --10  died       --10                                                                              died    --10                                                                               died                                          10               10         10                                         ______________________________________                                    

                  TABLE III                                                        ______________________________________                                         1 × 10.sup.2 L1210 Tumor Cells in                                        BDF.sub.1 Mice -                                                               Effects of Enkephalins                                                                 Methionine-            Leucine-                                                Enkephalin             Enkephalin                                      Days*   10 mg/kg s.c. Controls 10 mg/kg s.c.                                   ______________________________________                                         15      3 died        5 died   1 died                                          16      2 died        1 died   1 died                                          17      2 died        1 died   0 died                                          18      0 died        1 died   1 died                                          19      1 died        0 died   0 died                                          20      1 died        1 died   0 died                                          21      0 died        0 died    0 died**                                               9 died        9 died   3 died                                          ______________________________________                                          *Enkephalin injections started on day two                                      **Chi square test p < 0.025 between controls and leucineenkephalin       

With the high tumor cell count of 1×10⁴, no differences in survival times between controls, methionine-enkephalin, or leucine-enkephalin treated groups were seen (TABLE I). In the experiment in which the mice were inoculated with 1×10³ tumor cells (TABLE II), survival time for the controls was 16 days, 18 days for the methionine-enkephalin group, and 18 days for the leucine-enkephalin treated group. However, in the third experiment, carried out at 1×10² tumor cells, the survival time for the controls as well as the methionine-enkephalin treated group was 20 days, while the leucine-enkephalin group had a survival time of >20 days.

The seven surviving mice from experiment three were continued on daily leucine-enkephalin medication (10 mg/kg i.p.) until day 36. The mice did not receive any additional leucine-enkephalin medication on subsequent days of the experiment. On day 37, the surviving seven mice were rechallenged with an additional inoculum of tumor cells (1×10⁴ cells). One of the seven mice so treated died on day 52. The remaining six mice survived until day 64, at which time they were rechallenged further with a higher inoculum of tumor cells (1×10⁶). All six mice died by day 70. These rechallenge studies suggest that leucine-enkephalin is stimulating the immune system as measured by survival of the test animals.

EXAMPLE Ia

The previous third experiment with 1×10² L1210 tumor cells was repeated two more times to increase the total number of mice to thirty per group, thus enhancing the statistical reliability of the results. The composite results of the three runs are presented below in TABLE IIa. Again, a statistically significant difference determined by the Chi square test (p<0.05) was found between the leucine-enkephalin treated group and the control group corresponding to direct experimental evidence of extraordinary stimulation of the immunity systems.

                  TABLE IIa                                                        ______________________________________                                         1 × 10.sup.2 Tumor Cells                                                 in BDF.sub.1 Mice                                                                      Methionine-             Leucine-                                               Enkephalin              Enkephalin                                     Days    10 mg/kg s.c.                                                                              Controls    10 mg/kg s.c.                                  ______________________________________                                         14      1/30    Died    1/30  Died  --                                         15      10/30           12/30       5/30   Died                                16      18/30           21/30       13/30                                      17      25/30           24/30       15/30                                      18      25/30           25/30       16/30                                      19      26/30           26/30       --                                         20      27/30           27/30       --                                         21      27/30           27/30       --                                         Total   27/30           27/30       16/30**                                    ______________________________________                                          **Significant difference from controls p < 0.05 Chi square test.         

EXAMPLE Ib

The above experiments were repeated again using 1×10² L1210 tumor cells with 30 mg/kg dosage rate of methionine-enkephalin to a group of thirty mice. The results as presented in TABLE IIb confirmed a statistically significant difference between the methionine-enkephalin treated group and the control group at this high dosage rate.

                  TABLE IIb                                                        ______________________________________                                         1 × 10.sup.2 Tumor Cells                                                                       Methionine-                                                                    Enkephalin                                               Days       Controls   30 mg/kg                                                 ______________________________________                                         13         --          1                                                       14          2          4                                                       15         20         15                                                       16         35         20                                                       17         41         22                                                       18         43         23                                                       19         44         --                                                       20          0         --                                                       21          0         --                                                       22         --         24                                                                  44/49 Dead 24/30 Dead                                                          ED50 =     ED50 =                                                              15 ± 0.4 days                                                                          18 ± 1.2 days**                                       ______________________________________                                          **Significant difference from control p < 0.05 Chi square test.          

EXAMPLE II

A second set of three experiments was performed in a manner similar to the experiments of EXAMPLE I except that 25 mice were used as controls, 30 mice were treated with methionine-enkephalin and another 30 were treated with leucine-enkephalin. In each case the mice were inoculated with 1×10⁴ tumor cells and the enkephalin was administered daily at 30 mg/kg s.c. The data related to the survival of the mice are presented in TABLE IV.

                  TABLE IV                                                         ______________________________________                                         1 × 10.sup.4 L1210 Tumor Cells                                           in BDF.sub.1 Mice -                                                            Effects of Enkephalins*                                                                            30 mg/kg   30 mg/kg                                                            Methionine-                                                                               Leucine-                                        Days    Controls    Enkephalin Enkephalin                                      ______________________________________                                         # dead/# tested                                                                13       7/25        5/30       3/30                                           14      10/25        7/30       5/30                                           15      16/25        8/30      12/30                                           16      20/25       12/30      17/30                                           17      21/25       15/30      22/30                                           18      21/25       18/30      22/30                                           19      21/25       18/30      26/30                                           20      22/25       19/30      26/30                                           21      22/25       19/30      26/30                                           Total   22/25        19/30**   26/30                                           ______________________________________                                          *Pooled three experiments                                                      **Significant difference from controls p < 0.05 Chi square test          

As indicated in TABLE IV, approximately half the control mice died by the fourteenth day. In contrast, half of the methionine-enkephalin treated mice died by the seventeenth day. By day 21 of the experiment, eleven out of the thirty treated mice had survived. Delayed deaths were also seen in mice treated with leucine-enkephalin. The median day of death was about sixteen days. Thus, the last three experiments (EXAMPLE II) demonstrate that the higher doses of both leucine-and methionine-enkephalin (30 mg/kg) are effective in prolonging survival of mice inoculated with 1×10⁴ cells. Of great interest is the apparent potency of methionine-enkephalin, resulting in survival at day 21 of 11 of 30 mice. This survival finding may be related to and be consistent with the previous observation that specific receptor binding sites for the enkephalins are present on T lymphocytes.

A dramatic increase in survival time of the leucine-enkephalin treated mice in the third experiment of EXAMPLES I and Ia and the sharp contrast of these leucine-enkephalin treated mice when compared both to the control mice and to the methionine-enkephalin treated mice (9 out of 10 expired between days 14-20), can be seen when comparing the results of the third experiment to those of the first and second experiments of EXAMPLE I (TABLES I-III). This indicates that there is remarkable and sharp separation in the activities of leucine- versus methionine-enkephalin at lower inoculation rates (1×10² cells) and these effects persist even after rechallenging with tumor cells. This is again consistent with the original hypothesis and also suggests that leucine-enkephalin plays a neuroendocrine messenger role between the central nervous system and the immune system.

EXAMPLE III

In order to test for any synergistic effect of using an endogenous enkephalin with a known chemo-therapeutic agent, a study involving the inoculation of groups of mice with massive doses (1×10⁶) of L1210 tumor cells was performed in a manner analogous to the previous examples transferred in RPMI 1640 at pH 7. Three different concentrations of cis-platin, with and without methionine-enkephalin treatment, were administered and compared to a control involving neither cis-platin nor methionine-enkephalin. The following TABLES V, VI, and VII represent the results of this study. Statistical analysis of the data by the Chi square test confirms that cis-platin (the chemo-therapeutic agent) exhibits significant statistical delay of the rate of onset of mortality relative to the control and that the combination of methionine-enkephalin treatment with cis-platin exhibits a statistically significant delay in the mortality rate relative to the use of cis-platin by itself and that the significance of the statistical trend is enhanced as time progresses.

                  TABLE V                                                          ______________________________________                                         1 × 10.sup.6 L1210 Tumer Cells In                                        BDF.sub.1 Mice - Single Dose of                                                8 mg/Kg cis-platin                                                             30 mg/Kg s.c. Methionine-Enkephalin                                                 Cis-platin and     Saline                                                 Days Methionine-Enkephalin                                                                             Control   Cis-platin                                   ______________________________________                                          8                      10/20                                                   9                      19/20      2/20*                                       10                      20/20                                                  11                                                                             12    1/20*                        4/20*                                       13    3/20*                         7/20**                                     14    7/20*                        12/20**                                     15    13/20**                     16/20                                        16   16/20                        18/20                                        17   18/20                                                                     18                                19/20                                        19                                                                             20                                20/20                                        21   19/20                                                                          Median Day of Death ± s.e.                                             ED50           ED50      ED50                                                  16 day ±    8 days ±                                                                              12 Days ±                                          0.6            0.3       0.8                                                   ______________________________________                                          *Chi square test p < 0.05 significant difference                               **Chi square test p < 0.10                                               

                  TABLE VI                                                         ______________________________________                                         1 × 10.sup.6 Tumor Cells                                                 4 mg/Kg Cis-platin and                                                         30 mg/Kg s.c. Methionine-Enkephalin                                                  Cis-platin and     Saline                                                Days  Methionine-Enkephalin                                                                             Control   Cis-platin                                  ______________________________________                                          8                        8/20                                                  9                       17/20                                                 10     1/20              19/20                                                 11     6/20*             20/20      10/20*                                     12     13/20**                     15/20                                       13    19/20                        19/20                                       14                                                                             15    20/20                        20/20                                             Median Day of Death ± s.e.                                            ED50           ED50      ED50                                                  12 Days ±   8 Days ±                                                                              10 Days ±                                          0.5            0.3       0.5                                                   ______________________________________                                          Significant difference from controls                                           *Chi square test p < 0.05                                                      **Chi square test p < 0.10                                               

                  TABLE VII                                                        ______________________________________                                         1 × 10.sup.6 Tumor Cells                                                 2 mg/Kg Cis-platin and                                                         30 mg/Kg s.c. Methionine-Enkephalin                                                 Cis-platin and     Saline                                                 Days Methionine-Enkephalin                                                                             Control   Cis-platin                                   ______________________________________                                          8                       9/30                                                   9    1/20*             12/30      3/20*                                       10    6/20*             18/30      13/20*                                      11    11/20*            20/30      14/20*                                      12    14/20**           23/30      15/20**                                     13   16/20              25/30     17/20                                        14   18/20              29/30     19/20                                        15   20/20                                                                     16                      30/30                                                  17                                                                             18                                                                                  Median Day of Death ± s.e.                                             ED50           ED50      ED50                                                  11 Days ±   9 Days ±                                                                              10 Days ±                                          0.6            0.6       0.7                                                   ______________________________________                                          Significant difference from controls                                           *Chi square test p < 0.05                                                      **Chi square test p < 0.10                                               

EXAMPLE IV

In order to further demonstrate the immune system stimulating characteristics of leucine-enkephalin and methionine-enkephalin, a Phytohemagglutinin (PHA) stimulated mouse lymphocyte blastogenesis study was performed. The effects of methionine-and leucine-enkephalin on PHA stimulated lymphocyte transformation are presented in TABLES VIII and IX. The results presented in the study correlate well with previous mortality studies in mice inoculated with L1210 particularly with respect to the concentration dependence of methionine-and leucine-enkephalin stimulation effects as well as verify extraordinary immune system stimulation.

                                      TABLE VIII                                   __________________________________________________________________________     Methionine Enkephalin and Lymphocyte Blastogenesis                             (mean counts/minute ± s.e.)                                                         PHA (1:100)                                                                              PHA (1:250)                                                                               PHA (1:500)                                                                               PHA (1:750)                            __________________________________________________________________________     Controls                                                                               (11)                                                                              1458 ± 90                                                                          (11)                                                                               5840 ± 553                                                                         (11)                                                                               8737 ± 970                                                                         (10)                                                                               7686 ± 337                      1   mg/ml                                                                              (4)                                                                               4393 ± 664**                                                                       (4)                                                                               11807 ± 3526**                                                                      (4)                                                                               13405 ± 6755                                                                        (4)                                                                               10678 ± 5634                     10.sup.-1                                                                          mg/ml                                                                              (6)                                                                               5258 ± 990**                                                                       (6)                                                                               17378 ± 4352**                                                                      (6)                                                                               19321 ± 2684**                                                                      (6)                                                                               15791 ± 1829**                   10.sup.-2                                                                          mg/ml                                                                              (6)                                                                               3144 ± 500**                                                                       (6)                                                                               10516 ± 2838**                                                                      (6)                                                                               13794 ± 2855*                                                                       (6)                                                                               10779 ± 1018**                   10.sup.-4                                                                          mg/ml                                                                              (6)                                                                               2123 ± 228**                                                                       (6)                                                                                7971 ± 1113*                                                                       (6)                                                                               10470 ± 1967                                                                        (6)                                                                                9678 ± 1005**                   10.sup.-6                                                                          mg/ml                                                                              (6)                                                                               2357 ± 155**                                                                       (6)                                                                                7764 ± 912*                                                                        (6)                                                                               11208 ± 1941                                                                        (6)                                                                                9164 ± 394**                    10.sup.-8                                                                          mg/ml                                                                              (4)                                                                               1884 ±  188**                                                                      (4)                                                                                8764 ± 2693                                                                        (4)                                                                               11702 ± 3071                                                                        (4)                                                                                8348 ± 99                       10.sup.-10                                                                         mg/ml                                                                              (2)                                                                               1538 ± 170                                                                         (2)                                                                                4237 ± 473                                                                         (2)                                                                                6755 ± 742                                                                         (2)                                                                                8324 ± 244                      __________________________________________________________________________      *= p < 0.10 Significant difference from PHA controls                           **= p < 0.05 Significant difference from PHA controls                          () = Number of individual samples quantified in liquid scintillation           counter                                                                  

                                      TABLE IX                                     __________________________________________________________________________     Leucine Enkephalin and Lymphocyte Blastogenesis                                (mean counts/minute ± s.e.)                                                         PHA (1:100)                                                                              PHA (1:250)                                                                               PHA (1:500)                                                                               PHA (1:750)                            __________________________________________________________________________     Controls                                                                               (11)                                                                              1458 ± 90                                                                          (11)                                                                               5840 ± 553                                                                         (11)                                                                               8737 ± 970                                                                         (10)                                                                               7686 ± 337                      1   mg/ml                                                                              (4)                                                                               1114 ± 310                                                                         (4)                                                                                4411 ± 1945                                                                        (4)                                                                                6752 ± 1200                                                                        (4)                                                                               10589 ± 583*                     10.sup.-1                                                                          mg/ml                                                                              (6)                                                                               1516 ± 207                                                                         (6)                                                                                5265 ± 1076                                                                        (6)                                                                                9033 ± 356                                                                         (6)                                                                                8543 ± 1203                     10.sup.-2                                                                          mg/ml                                                                              (6)                                                                               1650 ± 180                                                                         (6)                                                                                6154 ± 370                                                                         (6)                                                                               10711 ± 1654                                                                        (6)                                                                               10468 ± 1117**                   10.sup.-4                                                                          mg/ml                                                                              (6)                                                                               1918 ± 293*                                                                        (6)                                                                                8273 ± 1586*                                                                       (6)                                                                               13429 ± 2885*                                                                       (6)                                                                                9213 ± 675**                    10.sup.-6                                                                          mg/ml                                                                              (6)                                                                               2425 ± 323**                                                                       (6)                                                                                8764 ± 727**                                                                       (6)                                                                               13092 ± 2505*                                                                       (6)                                                                               10814 ± 985**                    10.sup.- 8                                                                         mg/ml                                                                              (4)                                                                               2759 ± 387**                                                                       (3)                                                                               10710 ± 2142**                                                                      (4)                                                                               14052 ± 3403*                                                                       (4)                                                                                8719 ± 665                      10.sup.-10                                                                         mg/ml                                                                              (2)                                                                               2065 ± 360**                                                                       (2)                                                                                9825 ± 3575**                                                                      (2)                                                                                8074 ± 611                                                                         (2)                                                                                8559 ± 52;                      __________________________________________________________________________      *= p < 0.10 Significant Difference from PHA Controls                           **= p < 0.5 Significant Difference from PHA Controls                           () = Number of Individual Samples Quantified in Liquid Scintillation           Counter                                                                  

EXAMPLE V

In order to further demonstrate the cell mediated immunity system characteristics of the present invention, a series of in vitro tests were performed on human blood from normal volunteers. The study involved determining the active T-cell rosettes population (see further details of test procedure in Example VI) of the untreated blood and for treated blood at one of four concentrations of either leucine- or methionine-enkephalin addition as well as determining the total rosettes for both enkephalins at the four respective dosage levels. Data summarizing the study are presented in TABLES X through XIV. The data establishes that both leucine-enkephalin and methionine-enkephalin increased significantly the active T-cell rosettes of normal volunteers and that neither enkephalin alters total T-cell rosettes. It is concluded from the data that endogenous enkephalins play a role in cell mediated immunity and specifically T-cell function.

                  TABLE X                                                          ______________________________________                                         Methionine-Enkephalin - Percentages                                            of Active T-Cell Rosettes                                                                 Untreated                                                                              Methionine-Enkephalin                                       ______________________________________                                         10.sup.-2                                                                              mg/cc.   38.4 ± 5.6                                                                            (9/11) 51.8 ± 4.8                                10.sup.-6                                                                              mg/cc.   31.9 ± 4.6                                                                            (7/11) 47.9 ± 4.8*                               10.sup.-10                                                                             mg/cc.   34.1 ± 5.3                                                                            (9/11) 54.4 ± 3.9*                               10.sup.-14                                                                             mg/cc.   36.6 ± 7.7                                                                            (7/9) 48.3 ± 5.5                                 ______________________________________                                          *Significant difference from matching controls p < 0.05                        () = Number of samples in analyses with increased number of rosettes           compared to total number analyzed                                        

                  TABLE XI                                                         ______________________________________                                         Methionine-Enkephalin - Subjects with                                          Depressed Active T-Cell Rosettes                                                                            Methionine-                                               Untreated            Enkephalin                                        ______________________________________                                         10.sup.-2 mg/cc.                                                                         20        (2/11)         18                                                    27                       15                                          10.sup.-6 mg/cc.                                                                         20        (4/11)         14                                                    59                       30                                                    64                       34                                                    27                       25                                          10.sup.-10 mg/cc.                                                                        59        (2/11)         46                                                    27                       20                                          10.sup.-14 mg/cc.                                                                        31        (2/9)          24                                                    27                       12                                          Totals =            10/42   = 24%*                                             ______________________________________                                          () = Number of individual samples with depressed levels compared to total      number analyzed                                                                *No significant difference between untreated and methionineenkephalin          values                                                                   

                  TABLE XII                                                        ______________________________________                                         Leucine-Enkephalin - Percentages                                               of Active T-Cell Rosettes                                                                             Leucine-                                                            Untreated  Enkephalin                                              ______________________________________                                         10.sup.-2                                                                              mg/cc.    31.3 ± 4.8 (10/12)                                                                       47.4 ± 3.8*                                  10.sup.-6                                                                              mg/cc.    32.4 ± 4.6 (10/12)                                                                       45.9 ± 4.8                                   10.sup.-10                                                                             mg/cc.    34.0 ± 6.7 (8/12)                                                                        54.1 ± 5.1*                                  10.sup.-14                                                                             mg/cc.    32.7 ± 5.7 (9/12)                                                                        54.0 ± 4.2*                                              Totals = (37/48) = 77%                                             ______________________________________                                          *Significant difference from matching controls                                 () Number of samples used in analyses with matching controls             

                  TABLE XIII                                                       ______________________________________                                         Leucine-Enkephalin - Subject Samples                                           with Depressed Active T-Cell Rosettes                                                                        Leucine-                                                 Untreated             Enkephalin                                       ______________________________________                                         10.sup.-2 mg/cc.                                                                         31         (2/12)         12                                                   64                        33                                         10.sup.-6 mg/cc.                                                                         20         (2/12)         14                                                   64                        33                                         10.sup.-10 mg/cc.                                                                        31         (4/12)         25                                                   20                        10                                                   28                        11                                                   27                        15                                         10.sup.-14 mg/cc.                                                                        59         (3/12)         54                                                   28                        20                                                   27                        25                                         Total =              (11/48) = 23%*                                            ______________________________________                                          *No significant difference from controls p < 0.05                              () Number of depressed samples                                           

                  TABLE XIV                                                        ______________________________________                                         Total Rosettes                                                                 (Means ± s.e.)                                                                           Methionine-                                                                             Leucine-                                                              Enkephalin                                                                              Enkephalin                                               ______________________________________                                         Untreated          59.9 ± 3.1                                                                             59.9 ± 3.1                                    10.sup.-2                                                                              mg/cc.     59.9 ± 3.4                                                                             60.3 ± 3.3                                    10.sup.-6                                                                              mg/cc.     63.5 ± 2.7                                                                             58.0 ± 3.5                                    10.sup.-10                                                                             mg/cc.     58.0 ± 3.5                                                                             63.0 ± 2.4                                    10.sup.-14                                                                             mg/cc.     60.4 ± 3.4                                                                             60.9 ± 2.6                                    ______________________________________                                    

EXAMPLE VI

In a manner analogous to EXAMPLE V the active T-cell rosettes and total rosettes population of a series of human blood samples derived from lymphoma patients were measured. The clinical characteristics of the patients included in this study are outlined in TABLE XV. All patients had histologically diagnosed lymphoma, one with Hodgkin's disease and the remainder with nonHodgkin's lymphoma. The patients were considered to have advanced stages of disease at the time of study. One patient ("F") presented very bulky lymphadenopathy and was studied prior to and following the initiation of chemotherapy. 30 ml of heparinized blood was obtained from each of the patients and allowed to sediment. The leukocyte-rich plasma was collected and diluted twofold in phosphate-buffered saline (0.01M; pH 7.2). The diluted cell-rich plasma was layered over a Ficoll-Paque gradient cushion (Pharmacia, Piscataway, N.J.), centrifuged for 30 min at 400 g, the mononuclear band collected, washed twice, and resuspended in RPMI 1640 medium. The mononuclear cell fraction was evaluated for active and total T lymphocytes and the influence leucine-enkephalin and methionine-enkephalin had on the rosette process. The mononuclear cell fraction was first incubated with 25 μl of a 1/100 dilution of latex bead preparation/10⁶ cells for 30 min at 37° C. in order that any phagocytic cells would engulf the latex beads and thus be easily identified under the microscope. The cells were washed twice and adjusted to 1×10⁷ cells/ml in RPMI 1640 medium. A volume of 0.1 ml of cells was aliquoted into tubes along with 0.1 ml of varying dilutions of leucine-enkephalin and methionine-enkephalin incubated for 60 min at 37° C., and washed twice. The tubes designated for evaluation of active T cells received 0.1 m of RPMI 1640 medium to resuspend the pellet and 0.1 ml of fetal calf serum. The cells were allowed to incubate for another 60 min at 37° C., followed by the addition of 2×10⁷ sheep red blood cells. The cell suspension was centrifuged at 200 g for 5 min, gently resuspended, and visually quantitated on an hemocytometer. The tubes designated for evaluation of total T cells received 0.1 ml of RPMI 1640 medium to resuspend the pellet, 0.1 ml of fetal calf serum, and 2×10⁷ sheep red blood cells. The mixture was incubated at room temperature for 15 min, overnight at 4° C., and was returned to room temperature for 1 hr. The cell suspension was gently resuspended and visually quantitated on an hemocytometer. Any lymphocyte with three or more sheep red blood cells attached was considered a rosette. The results for the active or total T cells are reported as means and standard errors for the groups and statistical analyses were carried out by the Student t test. The enkephalins were purchased from CAL-MED, South San Francisco, Calif. (UCB Bioproducts, Brussels, Belgium) and reported to be chemically pure (>99%).

Significant increases in active rosettes were found when methionine-enkephalin (10⁻², 10⁻⁶, 10⁻¹⁰, and 10⁻¹⁴ mg/cc was added to lymphocytes taken from lymphoma patients (see TABLE XVI).

In sharp contrast to the positive enhancement effects of methionine-enkephalin, leucine-enkephalin was significantly effective only at one low concentration (10⁻¹⁴ mg/cc) and the actual percentages of active rosettes are shown in TABLE XVI. It is apparent that methionine-enkephalin (at all concentrations tested) elevates active rosette formation.

Neither methionine-enkephalin nor leucine-enkephalin, at any concentration tested, had any significant effect on levels of total rosettes (TABLE XVII).

Untreated patient levels of 39.2±5.1 (for methionine-enkephalin) as well as 36.4±3.9 (for leucine-enkephalin) were found to be significantly different from untreated normal volunteers (54.8±4.5%), a value which is taken from our continuing comparative studies in normal volunteers.

The study demonstrated a significant enhancement of active T-cell rosettes with methionine-enkephalin from lymphoma patients. Lymphoma patients are considered to be immunosuppressed--both by the nature of their disease and as a result of treatment. The fact that our patients were found to have significantly less total T-cell rosettes than normal volunteers supports this contention. Comparative data on active T-cell rosettes in 100 normal subjects were reported to be 28.4±6.5% when, on the other hand, patients with cancer had levels of active T-cell rosettes in the range of 8-24% (depending on the status of disease) (see Wybran and Fudenberg, J. Clin. Invest. 52, 1026, 1973). The present study shows that untreated patient levels of active T-cell rosettes (27.6 and 32.0%) did not differ significantly from normal volunteer levels (28.4±6.5%). EXAMPLE V indicates methionine-enkephalin increases active T-cell rosettes in blood from normal volunteers, and the data of EXAMPLE VI indicate similar findings that confirm enhancement of active rosettes in immunosuppressed patients.

The relative lack of enhancement of the active T-cell rosettes by leucine-ekephalin at higher concentrations and a significant enhancement of active T-cell rosettes at a very low concentration (10⁻¹⁴) suggests that specific enkephalin receptors (μ vs Δ) may depress or stimulate active rosette formation as a function of dosage.

Further, the reduction of immunocompetence resulting from prolonged stress, in part, may be a result of depletion of enkephalin stores. The present study does support the idea that "replacement therapy" with the endogenous ligand (methionine-enkephalin) or a closely related analog may be beneficial in restoring immunocompetence.

It is believed that the increase in active T-cell rosette-forming cells in our present study was a direct effect of nonmalignant population on T-cells because significant increases were seen in B-cell lymphoma and Hodgkin's disease.

Since the active T-cell rosette-forming cells represent a distinct subpopulation of T cells closely related to the overall level of cellular immunocompetence and since methionine-enkephalin is shown herein to enhance active T-cell rosettes in lymphoma patients, the present study postulates that immunomodulation by methionine-enkephalin provides a new approach to immunotherapy.

                                      TABLE XV                                     __________________________________________________________________________     Patient Characteristics                                                                                                Time of last treatment                 Patient                                                                            Age (years)                                                                          Diagnosis    Stage                                                                              Prior Therapy                                                                               prior to test (weeks)                  __________________________________________________________________________     A   51    B-cell lymphoma                                                                             IV  Splenectomy  52                                     B   83    Non-Hodgkin's lymphoma                                                                      III Cytoxan (Day 1)                                                                              4                                                                Vincristine (Day 1)                                                            Prednisone (Days 1-5)                               C   29    Hodgkin's    IIIB                                                                               MOPP →                                                                                8                                                                ABVO →                                                                  Total Nodal radiotherapy                            D   72    Non-Hodgkin's lymphoma                                                                      IV  Cytoxan (Day 1)                                                                             24                                                                Vincristine (Day 1)                                                            Adriamycin (Days 1-5)                                                          Prednisone (Days 1-5)                               E   45    Large-cell lyphoma                                                                          II  No therapy   --                                         45    Large-cell lymphoma                                                                         II  Cytoxan (Day 1)                                                                             3-4                                                               Vincristine (Day 1)                                                            Adriamycin (Day 1)                                                             Prednisone (Days 1-5)                               __________________________________________________________________________

                  TABLE XVI                                                        ______________________________________                                         Percentages of Active Rosettes                                                 After Incubation                                                                       Methionine-        Leucine-                                            Dose (mg)                                                                              Enkephalin         Enkephalin                                          ______________________________________                                         Untreated                                                                              27.6 ± 5.3      32.0 ± 4.1                                               (5 determinations) (8 determinations)                                  10.sup.-2                                                                            mg    46.6 ± 4.2*     41.1 ± 5.6                                   10.sup.-6                                                                            mg    49.0 ± 4.6*     42.5 ± 3.3                                   10.sup.-10                                                                           mg    46.8 ± 3.0*     40.3 ± 5.7                                   10.sup.-14                                                                           mg    48.5 ± 5.6*      52.7 ± 2.8*                                 ______________________________________                                          *Significant difference from untreated samples, p < 0.05                 

                  TABLE XVII                                                       ______________________________________                                         Total Rosettes                                                                              Methionine-   Leucine-                                                         Enkephalin    Enkephalin                                          Dose (mg)    percentages   percentages                                         ______________________________________                                         Untreated    39.2 ± 5.1 (5)                                                                            36.4 ± 3.9 (8)                                    10.sup.-2 mg                                                                               35.0 ± 6.9 32.6 ± 4.6                                        10.sup.-6 mg                                                                               42.2 ± 6.8 32.6 ± 5.6                                       10.sup.-10 mg                                                                               36.5 ± 6.5 37.6 ± 6.1                                       ______________________________________                                    

EXAMPLE VII

In order to illustrate the promoting of the natural immune system resisting growth of tumorous cells, a study of the effects of methionine-enkephalin and leucine-enkephalin on the so-called "natural killer" or NK cell activity was performed. A series of isolated human peripheral blood lymphocytes from various types of cancer patients as well as normal volunteers were incubated with enkephalin at one of four dosage levels and the NK activity was determined at 100:1, 33:1 and/or 11:1 effector-to-target cell ratios.

All blood samples utilized in this study were obtained from human volunteers. All experiments were performed with freshly isolated lymphocytes. Blood (approximately 30 ml) was collected by standard anticubital venipuncture and diluted 1:1 with RPMI 1640 medium. This mixture was layered over Histopaque (Sigma Chemical Co., St. Louis, MO) and centrifuged at 400 g for 30 minutes. The lymphocyte layer was removed, resuspended in RPMI 1640 and incubated in plastic petri dishes for one hour at 37° C. in 5% CO₂ in air to remove adherent cells (macrophages). The lymphocytes were then washed with RPMI 1640 and adjusted to a concentration of 1×10⁷ cells/ml. Viability was determined by trypan blue exclusion, and was always greater than 98 percent.

The K-562 tumor cell line, a myeloid cell line derived from a pleural effusion of a patient with a chronic myelocytic leukemia in blast crisis, was used as the target cell in these studies. The start for this cell line was supplied by the National Cancer Institute.

The NK cell assays were performed as previously described in literature (see deLandazuri et al, J. Immunology 1981). Aliquots containing 2×10⁶ target cells/ml were labeled with 150 μCi of ⁵¹ Cr solution/ml of cells (Amerisham, Arlington Heights, IL) by incubation at 37° C. in 5% CO₂ in air for 45 minutes. After washing the cells 3 times, 5×10³ viable cells in 0.1 ml of RPMI 1640 were pipetted into 96-well Linbro plates (Linbro Scientific, Hamden, CT). Various concentrations of effector cells in 0.1 ml of RPMI 1640 medium were added to triplate wells to give effector:target ratios of 100:1, 33:1 and 11:1. After incubation at 37° C. in 5% CO₂ in air for 4 hours 100 μl of supernatant from each well was collected and counted for 2 minutes in a Beckman 4000 autogamma counter. The percentage of isotope released was used as a measure of cytotoxity and was calculated by the following formula: ##EQU1## where: cpm experimental release=counts released after incubation of target cells with effector cells.

cpm from medium control=counts spontaneously released by target cells incubated in medium alone.

cpm maximum release=counts released by lysis of target cells with 1% Triton X-100.

Both methionine-enkephalin and leucine-enkephalin were purchased from Peninsula Laboratories, Inc. (San Carlos, CA) as synthesized, chemically pure preparations. Aliquots of peripheral blood lymphocytes prepared as above were incubated for one hour at 37° C. in 5% CO₂ in air with an equal volume of varying dilutions (10⁻⁴, 10⁻⁶, 10⁻⁸, 10⁻¹⁰, and 10⁻¹⁴ mg/ml) of either methionine-enkephalin or leucine-enkephalin. At the end of the incubation period the lymphocytes were washed with RPMI-1640 medium three times, resuspended in RPMI-1640 medium and utilized in NK cell assays.

The comparison of differences between treatment groups and non-treated cells was accomplished by the paired difference test.

The data corresponding to this in vitro study is presented in TABLES XVIII and XIX and FIGS. 1 through 4 of the drawing. The data presented in FIGS. 1 and 2 correspond to the normal volunteers while TABLES XVIII and XIX and FIGS. 3 and 4 correspond to cancer patients. The population of the volunteers used in the study fell into two groups with respect to spontaneous NK activity. Categorically the volunteers started from either a high initial NK activity or they had a relatively low initial NK activity. Thus the data presented in the FIGURES is analyzed as the population as a whole (legend A), the relatively low NK activity (legend B for FIGS. 1 and 2; legend C for FIGS. 3 and 4) and the relatively high NK activity (legend C for FIGS. 1 and 2; legend D for FIGS. 3 and 4) subgroups. The data illustrated in the FIGURES are from assays run at an effector-to-target cell ratio of 11:1 and are presented as percent increase in NK activity of treated cells above activity of non-treated cells from the same individual. In each case, the peripheral blood lymphocytes were treated with specified enkephalin at the concentrations shown for one hour and then assayed for NK activity. The stars indicate significant difference from nontreated cells (p<0.05).

As illustrated in FIG. 1, methionine-enkephalin enhanced NK cell activity of all concentrations tested. Ths enhancement was seen at all effector-to-target cell ratios investigated, but tended to be greatest in the 11:1 cell ratio. The enhancement induced by methionine-enkephalin tended to be biphasic with a dip in enhancement at the 10⁻⁸ mg/ml concentration. This tended to be the case with all individuals in the study and is reflected in the pooled data.

The observed percentage increase in NK activity following methionine-enkephalin treatment was higher in individuals with a naturally low NK activity, but only significant at one concetration (10⁻⁶ mg/ml), while the percentage increase in activity was significant at three concentrations (10⁻⁶, 10⁻⁸, and 10⁻¹⁰ mg/ml) in individuals with naturally high NK activity.

Treatment with leucine-enkephalin (FIG. 2) enhanced NK activity similarly to methionine-enkephalin. However, leucine-enkephalin was more active than methionine-enkephalin as evidenced by much greater percentage increases in NK activity following treatment with leucine-enkephalin. NK activity was significantly enhanced across the range of leucine-enkephalin concentrations in low activity individuals while only the three lowest concentrations induced significant changes in the high activity individuals.

As presented in TABLES XVIII and XIX and as illustrated in FIGS. 3 and 4, methionine-enkephalin as well as leucine-enkephalin enhanced NK cell activity in peripheral blood lymphocytes isolated from cancer patients in a manner analogous to the normal volunteers. Both are effective at all concentrations tested and at all effector-to-target cell ratios investigated. The biphasic dip in enhancement for methionine-enkephalin appeared at 10⁻¹⁰ mg/ml while a suggestion of biphasic behavior at 10⁻⁸ mg/ml for leucine-enkephalin was present. Again, the observed percentage increase in NK activity following treatment was higher in individuals with a natural low NK activity (see data of TABLE XIX and legend B of FIGS. 3 and 4), while the percentage increase in activity was significant at all concentrations for methionine-enkephalin and two concentrations for leucine-enkephalin (10⁻⁸ and 10⁻¹⁰ mg/ml) in individuals with low NK activity and was significant at only one concentration (10⁻¹⁰ mg/ml) for methionine-enkephalin and two concentrations (10⁻⁶ and 10⁻⁸) for leucine-enkephalin in individuals with high NK activity.

The results of this study confirm the previous conclusion that enkephalins are immunostimulatory. Perhaps of special interest is the observation that those normal individuals with relatively low natural NK activity exhibited the largest increase in NK activity with enkephalin treatment. This is similar to the observation made with active T-cell rosettes in lymphoma patients and normal individuals. Except in contrast to normal volunteers, lymphoma patients have low numbers of T-rosette forming cells and showed the largest increase in T-cell rosettes with enkephalin treatment. Also, there are apparent differences in the potential of enkephalins in their effects on various immune functions. Leucine-enkephalin is observed to be much more active in stimulating NK activity than methionine-enkephalin.

In contrast, previous EXAMPLES with lymphocytes from normal human volunteers as well as lymphoma patients suggest that methionine-enkephalin is more active than leucine-enkephalin in enhancing active T-cell rosettes. In addition, the two enkephalins exhibited potency differences in mouse splenic lymphocyte blastogenesis induced by phytohemagglutinin. Leucine-enkephalin was active at low concentrations and inactive at higher levels while methionine-enkephalin behaved in an opposite manner. These apparent differences in activity or potency of the enkephalins again suggest major differences in affinities for cellular receptors.

In view of the data it is further proposed that the enkephalins play a role in a central nervous system-endocrine-immune interrelationship in which dysfunctions in the immune system may be chronic stress related and be accompanied by perturbations in enkephalin levels in the adrenals, the primary source of enkephalins in peripheral circulation. Large amounts of enkephalins, catecholamines and steroids are secreted simultaneously from the adrenals during stress. It is well known that the central nervous system and behavior can influence immune function. Recently behavioral states have been shown to influence cerebral spinal fluid levels of endorphins and exercise has been shown to increase plasma levels of endorphins. Exercise has also been shown to enhance human NK activity. Due to these facts and the effects of enkephalins referred to above, it is felt that the enkephalins play a central role in natural immunomodulation which is influenced by a number of factors; i.e., the enkephalins are immunomodulators.

                                      TABLE XVIII                                  __________________________________________________________________________     Patient                                                                            Age                                                                               Sex                                                                               R.sub.x     Stage   R.sub.x Prior to Study                           __________________________________________________________________________     1   59 F  Thyroid Cancer                                                                             Advanced, IV                                                                           Adriamycin 1 month prior study                   2   48 F  Acute Myelocytic                                                                           Bone marrow                                                                            Induction R.sub.x, with Adria-                             Leukemia            mycin, Prednisone, Vincristine,                                                ARAC                                             3   73 F  Small Cell Cancer                                                                          Extensive                                                                              VP16 1 week before study                                   Lung, Relapse                                                        4   30 F  Hodgkin's Recurrent                                                                        Stage IV                                                                               Multiple chemotherapy. MOPP                                                    ABVD. Radiation R.sub.x. Last                                                  R.sub.x was 1 month prior to                                                   study using methyl GaG.                          5   49 F  Chronic Myeologenous                                                                       Lymphoblastic                                                                          Vincristine/Prednisone during                              Leukemia    Transformation                                                                         study                                            6   48 F  Breast Cancer Stage                                                                        Stage V On Chemotherapy 5Fu, L-pam                       7   50 F  Ovarian Cancer                                                                             Stage III                                                                              Adriamycin, Cytoxan, Cisplatinium.               8   72 M  Gastric Carcinoma                                                                          Advanced                                                                               5Fu. Adriamycin, Mitomycin C,                                                  Radiation R.sub.x.                               9   90 M  Poorly differentiated,                                                                     IV      No chemotherapy for several                                lymphocytic lymphoma,                                                                              months, prior to that received                             modular             C-M Opp.                                         10                                                                             11  46 F  Diffuse Histiocytic                                                                        III     CHOP. Radiation to abdomen.                                Lymphoma            COP-BLAM. 2 weeks prior to study.                12        Modular Histiocytic                                                                        IV      CHOP-Chemotherapy 1 month prior                            Lymphoma            to study. 7 courses given up                                                   to date of study.                                __________________________________________________________________________

                  TABLE XIX                                                        ______________________________________                                         Effect of Enkephalin on NK Cells Activity                                             100:1                                                                               %↑↓                                                                       33:1    %↑↓                                                                      11:1 %↑↓                       ______________________________________                                         Patient 1                                                                      Control  23.99          10.70        3.61                                      Methionine-                                                                    Enkephalin                                                                     10.sup.-6 mg/ml                                                                         28.80  20.05↑                                                                           12.96 21.12↑                                                                          5.26 45.71↑                         10.sup.-8                                                                               30.50  27.14↑                                                                           13.20 23.36↑                                                                          4.70 30.19↑                         10.sup.-10                                                                              30.39  26.68↑                                                                           10.79 0.89↑                                                                           4.28 18.56↑                         10.sup.-14                                                                              30.76  29.05↑                                                                           13.28 24.11↑                                                                          4.41 22.16↑                         Leucine-                                                                       Enkephalin                                                                     10.sup.-6                                                                               30.40  26.72↑                                                                           11.55 7.94↑                                                                           4.41 22.16↑                         10.sup.-8                                                                               28.37  18.26↑                                                                           10.29 3.83↓                                                                          4.44 22.99↑                         10.sup.-10                                                                              27.11  13.01↑                                                                           9.79  8.50↓                                                                          4.26 18.01↑                         10.sup.-14                                                                              28.32  18.05↑                                                                           10.34 3.36↓                                                                          3.73 3.32↑                          Patient 2                                                                      Control  9.52           2.12         0.78                                      Methionine-                                                                    Enkephalin                                                                     10.sup.-6                                                                               10.51  12.40↑                                                                           2.89  36.32↑                                                                          1.96 151.28↑                        10.sup.-8                                                                               10.04  5.46↑                                                                            3.57  68.40↑                                                                          1.46 87.18↑                         10.sup.-10                                                                              9.78   2.73↑                                                                            2.91  37.26↑                                                                          1.27 62.82↑                         10.sup.-14                                                                              --     --      3.23  52.36↑                                                                          1.72 120.51↑                        Leucine-                                                                       Enkephalin                                                                     10.sup.-6                                                                               --     --      4.66  119.81↑                                                                         1.79 129.49↑                        10.sup.-8                                                                               --     --      2.95  39.15↑                                                                          1.79 129.49↑                        10.sup.-10                                                                              --     --      2.86  34.91↑                                                                          1.26 61.54↑                         10.sup.-14                                                                              --     --      3.10  46.23↑                                                                          0.95 21.79↑                         Patient 3                                                                      Control  --             19.95        17.76                                     Methionine-                                                                    Enkephalin                                                                     10.sup.-6 mg/ml                                                                         --             --           --                                        10.sup.-8                                                                               --             --           --                                        10.sup.-10                                                                              --             22.33        17.74                                     10.sup.-14                                                                              --             25.48        24.01                                     Leucine-                                                                       Enkephalin                                                                     10.sup.-6                                                                               --             --           --                                        10.sup.-8                                                                               --             --           --                                        10.sup.-10                                                                              --             25.28        20.27                                     10.sup.-14                                                                              --             27.03        22.74                                     Patient 4                                                                      Control                 17.02        7.62                                      Methionine-                                                                    Enkephalin                                                                     10.sup.-6                                                                               --             18.77        8.62                                      10.sup.-8                                                                               --             21.60        9.42                                      10.sup.-10                                                                              --             21.97        9.04                                      10.sup.-14                                                                              --             23.45        10.09                                     Leucine-                                                                       Enkephalin                                                                     10.sup.-6                                                                               --             23.66        10.86                                     10.sup.-8                                                                               --             18.27        10.17                                     10.sup.-10                                                                              --             24.56        12.75                                     10.sup.-14                                                                              --             22.17        12.06                                     Patient 5                                                                      Control  1.00           0.812        0.247                                     Methionine-                                                                    Enkephalin                                                                     10.sup.-6 mg/ml                                                                         1.73           1.19         0.545                                     10.sup.-8                                                                               1.98           1.09         0.693                                     10.sup.-10                                                                              2.49           1.18         0.7                                       10.sup.-14                                                                              2.53           1.305        0.141                                     Leucine-                                                                       Enkephalin                                                                     10.sup.-6                                                                               1.56           1.28         0.493                                     10.sup.-8                                                                               2.57           1.04         0.765                                     10.sup.-10                                                                              2.34           1.32         1.241                                     10.sup.-14                                                                              1.68           1.09         0.752                                     Patient 6                                                                      Control                 6.50  --     3.85 --                                   Methionine-                                                                    Enkephalin                                                                     10.sup.-6               8.76  34.77↑                                                                          3.69 4.16↓                         10.sup.-8               8.75  34.62↑                                                                          4.01 9.35↑                          10.sup.-10              7.58  16.62↑                                                                          4.57 18.70↑                         10.sup.-14              8.40  29.23↑                                                                          4.15 7.79↑                          Leucine-                                                                       Enkephalin                                                                     10.sup.-6               8.79  35.23↑                                                                          3.44 10.65↓                        10.sup.-8               7.51  15.54↑                                                                          4.13 7.27↑                          10.sup.-10              8.78  35.08↑                                                                          5.07 31.69↑                         10.sup.-14              7.73  18.90↑                                                                          5.26 36.62↑                         Patient 7                                                                      Control                 33.44 --     16.54                                                                               --                                   Methionine-                                                                    Enkephalin                                                                     10.sup.-6               37.67 12.65↑                                                                          20.78                                                                               25.63↑                         10.sup.-8               40.65 21.56↑                                                                          19.86                                                                               20.17↑                         10.sup.-10              40.20 20.22↑                                                                          17.94                                                                               8.46↑                          10.sup.-14              40.87 22.22↑                                                                          19.46                                                                               17.65↑                         Leucine-                                                                       Enkephalin                                                                     10.sup.-6               38.07 13.85↑                                                                          16.76                                                                               1.33↑                          10.sup.-8               36.34 8.67↑                                                                           19.61                                                                               18.56↑                         10.sup.-10              38.44 14.95↑                                                                          16.85                                                                               1.87↑                          10.sup.-14              34.99 4.64↑                                                                           17.85                                                                               7.92↑                          Patient 8                                                                      Control                 14.59 --     8.59 --                                   Methionine-                                                                    Enkephalin                                                                     10.sup.-6               17.39 19.19↑                                                                          9.20 7.10↑                          10.sup.-8               16.61 13.85↑                                                                          9.67 12.57↑                         10.sup.-10              16.53 13.30↑                                                                          10.19                                                                               18.63↑                         10.sup.-14              16.39 12.34↑                                                                          9.25 7.68↑                          Leucine-                                                                       Enkephalin                                                                     10.sup.-6               15.63 7.13↑                                                                           10.03                                                                               16.76↑                         10.sup.-8               16.33 11.93↑                                                                          8.72 1.51↑                          10.sup.-10              14.87 1.92↑                                                                           9.03 5.12↑                          10.sup.-14              17.23 18.09↑                                                                          9.63 12.11↑                         Patient 9                                                                      Control                 48.37        30.25                                     Methionine-                                                                    Enkephalin                                                                     10.sup.-6               41.60        28.20                                     10.sup.-8               46.93        31.58                                     10.sup.-10              47.41        29.72                                     10.sup.-14              50.47        33.15                                     Leucine-                                                                       Enkephalin                                                                     10.sup.-6               53.80        34.58                                     10.sup.-8               53.99        33.67                                     10.sup.-10              49.81        32.74                                     10.sup.-14              49.44        32.69                                     Patient 10                                                                     Control  5.99           3.85         3.11                                      Methionine-                                                                    Enkephalin                                                                     10.sup.-6                                                                               10.07          3.96         1.51                                      10.sup.-8                                                                               11.12          5.53         2.18                                      10.sup.-10                                                                              13.36          6.00         3.14                                      10.sup.-14                                                                              12.68          4.57         2.38                                      Leucine-                                                                       Enkephalin                                                                     10.sup.-6                                                                               14 32          4.86         2.73                                      10.sup.-8                                                                               11.45          6.32         2.52                                      10.sup.-10                                                                              12.50          4.55         2.72                                      10.sup.-14                                                                              10.94          3.48         1.54                                      Patient 11                                                                     Control                 16.17        8.57                                      Methionine-                                                                    Enkephalin                                                                     10.sup.-6                                                                      10.sup.-8                                                                      10.sup.-10                                                                     10.sup.-14              14.99        8.93                                      Enkephalin                                                                     10.sup.-6                                                                      10.sup.-10                                                                     10.sup.-14              13.05        9.08                                      Patient 12                                                                     Control  12.47  --      6.71  --     3.37 --                                   Metionine-                                                                     Enkephalin                                                                     10.sup.-6                                                                               13.    6.20↑                                                                            6.84  1.94↑                                                                           3.73 16.62↑                         10.sup.-8                                                                               13.27  11.23↑                                                                           7.19  7.15↑                                                                           4.20 24.63↑                         10.sup.-10                                                                              13.66  9.54↑                                                                            6.77  3.87↑                                                                           4.34 28.78↑                         10.sup.-14                                                                              14.36  15.11↑                                                                           7.73  15.2↑                                                                           4.23 25.52↑                         Leucine-                                                                       Enkephalin                                                                     10.sup.-6                                                                               15.02  20.45↑                                                                           8.60  28.17  4.44 31.75↑                         10.sup.-8                                                                               14.50  16.28↑                                                                           7.94  18.33  4.27 26.71↑                         10.sup.-10                                                                              16.06  28.79↑                                                                           8.59  28.02  4.75 46.88↑                         10.sup.-14                                                                              14.72  18.04↑                                                                           9.49  41.43  5.58 65.58↑                         ______________________________________                                    

In order to demonstrate, in vivo, the highly positive stimulatory effects of methionine-enkephalin on T cells and NK cells when infused directly into the blood stream, clinical testing on normal human volunteers was performed using methods of analysis analogous to the previous examples. All prospective volunteers were screened and found to be normal in physical examination as well as clinical laboratory analysis. No candidates were admitted to the study with any medical history and candidates were particularly screened to be allergy free. The screened volunteers were admitted to the hospital the evening before the study. The next morning intravenous infusion of methionine-enkephalin was started at 9:00 a.m. for 30 minutes. Blood examples and vital signs were taken at appropriate intervals. The following examples summarize the results of the respective volunteers.

EXAMPLE VIII

The first volunteer received 10 micrograms/kg of methionine-enkephalin per kilogram of body weight. The level of active T-cell rosette cells increased 183% at 2 hours after infusion and 103% after 24 hours. The level of total T-cell rosettes also increased 98% at 2 hours and 82% at 24 hours over baseline control levels. In contrast, the NK cell levels were found to be reduced 24% at 2 hours and 56% at 24 hours after infusion (characteristic of normal volunteers at high control levels). No significant changes were seen in the blood pressure, temperature or EKG. Slight increase in heart rate and respiration were recorded. Neurological evaluations indicated slight nystagmus 1 to 2 hours after infusion. The 100 mm line test for behavioral effects indicated a shift in the score to the "happy" side for the rest of the study. The following TABLES XX through XXIV summarize the results of Volunteer No. 1.

                  TABLE XX                                                         ______________________________________                                         Methionine-Enkephalin (0.010 mg/kg)                                                    Blood       Heart    Resp.                                                     Pressure    Rate     Rate    Temp.                                     ______________________________________                                         First Day                   S/E                                                 8 a.m.   100/70    100/70  44/45  12    98 F.                                 8:55      100/70    100/70  50/52  13    98.8                                  9:00      METHIONINE-ENKEPHALIN INFUSION                                       9:10      100/70            60     14    98.8                                  9:20      100/70            60     12    98.8                                  9:30      100/70            60     12    98.8                                  10 a.m.   110/70            64     12    98.8                                  11 a.m.   100/70            60     14    98.6                                  1 p.m.    100/70    100/70  45/60  12    98.4                                  4 p.m.    100/70    100/70  44/56  13    98.4                                  Second Day                                                                     8:55      100/70    100/70  45/60  12    98.4                                  ______________________________________                                    

                  TABLE XXI                                                        ______________________________________                                         100 mm. LINE TEST                                                              DOSE - 10 micrograms per kg.                                                   ______________________________________                                         First Day                                                                      8:30       a.m.    55.0 mm.                                                    8:55       a.m.    59.0 mm.                                                    9:30       a.m.    55.0 mm.                                                    10:00      a.m.*   38.0 mm.                                                    11:00      a.m.*   38.0 mm.                                                    1:00       p.m.*   43.0 mm.                                                    4:00       p.m.*   40.0 mm.                                                    Next Day                                                                       8:55       a.m.    39.0 mm.                                                    ______________________________________                                    

                  TABLE XXII                                                       ______________________________________                                         Methionine-Enkephalin (0.01 mg/kg)                                             Active T-Cell Rosettes                                                                  MEAN VALUE                                                            ______________________________________                                         CONTROLS   30/200                                                               2 hours   85/200        183% INCREASE*                                        24 hours   61/200        103% INCREASE*                                        ______________________________________                                    

                  TABLE XXIII                                                      ______________________________________                                         Methionine-Enkephalin (0.01 mg/kg)                                             Total T-Cell Rosettes                                                                   MEAN VALUE                                                            ______________________________________                                         CONTROLS    62/200                                                              2 hours   123/200       98% INCREASE*                                         24 hours   113/200       82% INCREASE*                                         ______________________________________                                    

                  TABLE XXIV                                                       ______________________________________                                         NK Cell Activity                                                               Methionine-Enkephalin (0.01 mg/kg)                                             RATIO 33:1                                                                              % ACTIVITY                                                            ______________________________________                                         CONTROLS   51%                                                                  2 HOURS   39%          24% DECREASE                                           24 HOURS   23%          56% DECREASE                                           ______________________________________                                    

EXAMPLE IX

In a manner analogous to Volunteer No. 1 of EXAMPLE VIII, Volunteer No. 2 received 10 micrograms of methionine-enkephalin per kilogram of body weight. The volunteer showed significant increases of active T-cell rosettes of 126% in 2 hours and 26% at 24 hours after infushion. Only slight increases were seen in the total T-cell rosettes. The NK cell levels showed a slight decrease (10% at 2 hours) but a highly significant increase at 33% at 24 hours. No changes were seen in blood pressure, body temperature, or EKG. Slight decreases in heart rate were seen in the afternoon following the infusion. Neurological evaluation indicated a slight nystagmus 2 hours after infusion. And again, a slight shift towards the "happy" side was seen in the 100 millimeter line test score. The following TABLES XXV-XXX summarize the results of in vivo testing of Volunteer No. 2.

                  TABLE XXV                                                        ______________________________________                                         Methionine-Enkephalin (0.010 mg/kg)                                                   Blood       Heart    Resp.                                                     Pressure    Rate     Rate     Temp.                                     ______________________________________                                         First Day                  S/E                                                 8 a.m.   130/80    130/80  72/80  12     98 F.                                 8:55     130/80    130/87  70/76  12     98                                    9:00     Methionine-Enkephalin Infusion                                        9:10     130/80            70     10     98                                    9:20     130/80            62     12     98                                    9:30     130/80            72     12     98                                    10:00    130/80            76     12     98                                    11:00    130/80    130/80  66/70  13     98                                    1 p.m.   130/80    130/80  64/70  13     98                                    4 p.m.   130/80    140/80  68/72  12     98                                    Next Day                                                                       8:44     130/80    130/80  64/72  12     98                                    ______________________________________                                    

                  TABLE XXVI                                                       ______________________________________                                         100 mm. Line Test                                                              DOSE = 10 micrograms per kg.                                                   ______________________________________                                         First Day                                                                      8:00       a.m.   49.0 mm.                                                     8:55       a.m.   46.0 mm.                                                     9:30       a.m.*  44.0 mm.                                                     10:00      a.m.   47.0 mm.                                                     11:00      a.m.   50.0 mm.                                                     1:00       p.m.   49.0 mm.                                                     4:00       p.m.   50.0 mm.                                                     Next Day                                                                       8:55       a.m.*  44.0 mm.                                                     ______________________________________                                    

                  TABLE XXVII                                                      ______________________________________                                         Methionine-Enkephalin (0.01 mg/kg)                                             Active T-Cell Rosettes                                                                   MEAN VALUE                                                           ______________________________________                                         CONTROLS    34/200                                                              2 hours    77/200        126% Increase                                        24 hours    43/200         26% Increase                                        ______________________________________                                    

                  TABLE XXVIII                                                     ______________________________________                                         Methionine-Enkephalin (0.01 mg/kg)                                             Total T-Cell Rosettes                                                                    MEAN VALUE                                                           ______________________________________                                         CONTROLS    113/20                                                              2 hours    117/200        4% Increase                                         24 hours    123/200        9% Increase                                         ______________________________________                                    

                  TABLE XXIX                                                       ______________________________________                                         NK CELLS                                                                       11:1 RATIO                                                                     Methionine-Enkephalin (0.010 mg/kg)                                            ______________________________________                                         CONTROLS - 19%                                                                 2 hours - 17%        10% Decrease                                              24 hours - 25%*      33% Increase                                              ______________________________________                                    

                                      TABLE XXX                                    __________________________________________________________________________     Methionine-Enkephalin (0.01 mg/kg)                                             NK CELL ACTIVITY                                                                      RATIO 33:1       RATIO 11:1                                                    % ACTIVITY       % ACTIVITY                                             __________________________________________________________________________     CONTROLS                                                                              38%              19%                                                     2 hours                                                                              36%      6% Decrease                                                                            17%     10% Decrease                                   24 hours                                                                              32%     16% Decrease                                                                            25%     33% Increase                                   __________________________________________________________________________

EXAMPLE X

In view of the composite of the results associated with Volunteer No. 1 and No. 2, a third volunteer was administered a smaller dose of methionine-enkephalin (0.001 mg/kg) corresponding closer to the original in vitro test concentrations of methionine-enkephalin. Significant increases in active T-cell rosettes were seen at 2 and 24 hours of 71% and 53%, respectively, over control levels. Essentially no change in total T-cell rosettes was observed. Significant increases (15% and 30%) in the level of NK cells were seen at 2 and 24 hours after infusion of the methionine-enkephalin. Slight decreases in heart rate and respiratory rate were recorded at the end of the infusion. No other significant changes were detected. Slight nystagmus as well as a slight shift on the 100 millimeter line test to the "happy" side was recorded following the infusion. The following TABLES XXXI through XXXVI summarize the results of Volunteer No. 3.

                  TABLE XXXI                                                       ______________________________________                                         Methionine-Enkephalin (0.001 mg/kg)                                                   Blood       Heart   Resp.                                                      Pressure    Rate    Rate     Temp.                                      ______________________________________                                         First Day                  S/E                                                 8 a.m.   110/80   110/80   52/60 12     98° F.                          8:30     110/80   110/80   56/60 12     98                                     8:55     110/80   110/80   52/60 12     98                                     9:00     Methionine-Enkephalin Infusion                                        9:10     110/80            55    12     98                                     9:20     110/80            55    12     98                                     9:30     110/80            50    12     98                                     10:00    110/80            54    10     98                                     11:00    110/80   120/80   56/62 12     98                                     1 p.m.   110/80   110/80   55/58 12     98                                     4 p.m.   110/80   110/80   55/58 12     98                                     Next Day                                                                       8:55     110/80   120/80   54/60 12     98                                     ______________________________________                                    

                  TABLE XXXII                                                      ______________________________________                                         100 mm. LINE TEST                                                              DOSE = one microgram per kg.                                                   ______________________________________                                         8:00       a.m.   23.0 mm.                                                     8:30       a.m.   39.0 mm.                                                     8:55       a.m.   37.0 mm.                                                     9:30       a.m.   41.0 mm.                                                     10:00      a.m.*  28.0 mm.                                                     11:00      a.m.*  36.0 mm.                                                     1:00       p.m.   35.0 mm.                                                     4:00       p.m.   39.0 mm.                                                     ______________________________________                                    

                  TABLE XXXIII                                                     ______________________________________                                         Methionine-Enkephalin (0.001 mg/kg)                                            Active T-cell Rosettes                                                                   MEAN VALUE                                                           ______________________________________                                         CONTROLS    17/200                                                              2 hours    29/200         71% Increase                                        24 hours    26/200         53% Increase                                        ______________________________________                                    

                  TABLE XXXIV                                                      ______________________________________                                         Methionine-Enkephalin (0.001 mg/kg)                                            Total T-Cell Rosettes                                                                    MEAN VALUE                                                           ______________________________________                                         CONTROLS    100/200                                                             2 hours    106/200        6% Increase                                         24 hours    108/200        8% Increase                                         ______________________________________                                    

                  TABLE XXXV                                                       ______________________________________                                         Methionine-Enkephalin (0.001 mg/kg)                                            NK Cell Activity (33:1)                                                                  % Activity                                                           ______________________________________                                         CONTROLS    39%                                                                 2 hours    36%            8% Decrease                                         24 hours    50%           28% Increase                                         ______________________________________                                    

                  TABLE XXXVI                                                      ______________________________________                                         Methionine-Enkephalin (0.001 mg/kg)                                            11:1 RATIO                                                                     ______________________________________                                         CONTROLS    20%                                                                 2 hours    24%            15% Increase                                        24 hours    27%            30% Increase                                        ______________________________________                                    

EXAMPLE XI

In a manner analogous to Volunteer No. 3 of EXAMPLE IV, Volunteer No. 4 received 0.001 mg/kg of methionine-enkephalin. Volunteer No. 4 showed a significant increase of active T-cell rosettes of 148% in 2 hours and a decrease of 45% after 24 hours from infusion. Again, no significant change in total T-cell rosettes took place. Analogous to Volunteer No. 1 of EXAMPLE VIII, the NK cell levels were found to be reduced 30% at 2 hours and 34% at 24 hours relative to a high control level of a healthy normal volunteer. All other measurements and values were essentially equivalent to the previous volunteers as exhibited in the following TABLES XXVII through XXXVII.

                  TABLE XXXVII                                                     ______________________________________                                         DOSE = one microgram per kg.                                                          Blood      Heart    Resp.                                                      Pressure   Rate     Rate     Temp.                                      ______________________________________                                         First Day                                                                      8:00 a.m.                                                                               110/80   110/80  60/64  12/min.                                                                               98.2° F.                        8:55     110/80   110/80  60/64  12     98.1                                   9:00     Methionine-Enkephalin Infusion                                        9:10     110/80           63     13     98.2                                   9:20     100/80           67     12     98.2                                   9:30     100/80           57     12     98.2                                   10:00    110/80           74     10     98.2                                   11:00    100/80   100/80  66     12     98                                     1:00 p.m.                                                                               110/70   110/70  60/64  11     98                                     4:00     110/70   110/70  58/62  12     98                                     Next Day                                                                       8:55 a.m.                                                                               120/80   120/80  75/78  12     97                                     ______________________________________                                    

                  TABLE XXVIII                                                     ______________________________________                                         100 mm. Line Test                                                              Dose = one microgram per kg.                                                   ______________________________________                                         First Day                                                                      7:30        a.m.   26.0 mm.                                                    8:30               45.0 mm.                                                    8:55               29.0 mm.                                                    10:00*             26.0 mm.                                                    11:00              32.0 mm.                                                    1:00        p.m.   32.0 mm.                                                    4:00               38.0 mm.                                                    Second Day                                                                     8:55        a.m.   292.0 mm.                                                   ______________________________________                                    

                  TABLE XXXIX                                                      ______________________________________                                         Methionine-Enkephalin (0.001 mg/kg)                                            Active T-cell Rosettes                                                                 MEAN VALUE                                                             ______________________________________                                         Controls  31/200                                                                2 hours  77/200          148% Increase                                        24 hours  17/200           45% Decrease                                        ______________________________________                                    

                  TABLE XL                                                         ______________________________________                                         Methionine-Enkephalin (0.001 mg/kg)                                            Total T-Cell Rosettes                                                                  MEAN VALUE                                                             ______________________________________                                         Controls  116/200                                                               2 hours  124/200          4% Increase                                         24 hours  110/200          3% Decrease                                         ______________________________________                                    

                  TABLE XLI                                                        ______________________________________                                         Methionine-Enkephalin (0.001 mg/kg)                                            NK Cell Activity                                                               ______________________________________                                         100:1                                                                          Controls  59%                                                                   2 hours  49%            17% Decrease                                          24 hours  44%            25% Decrease                                          33:1                                                                           Controls  31%                                                                   2 hours  22%            30% Decrease                                          24 hours  21%            34% Decrease                                          ______________________________________                                    

EXAMPLE XII

In a manner analogous to the previous volunteers, Volunteers No. 5 and 6 were administered dosages of 50 micrograms of methionine-enkephalin per kilogram of body weight. The resulting active T-cell rosettes, total T-cell rosettes, and NK cell analysis at effector-to-target ratios are presented in TABLES XIII through XLII.

                  TABLE XLII                                                       ______________________________________                                         Dose = Fifty Micrograms/kg.                                                    Active T-cell Rosettes                                                                       No. 5 No. 6                                                      ______________________________________                                         Controls        17%     3%                                                      2 hours        41%     7%                                                     24 hours         8%     22%                                                    ______________________________________                                    

                  TABLE XLIII                                                      ______________________________________                                         Dose = Fifty Micrograms/kg                                                     Total T-Cell Rosettes                                                                         #5   #6                                                         ______________________________________                                         Controls         48%    59%                                                     2 hours         64%    63%                                                    24 hours         59%    65%                                                    ______________________________________                                    

                  TABLE XLIV                                                       ______________________________________                                         NK Cells                                                                       33:1                                                                                     #5               #6                                                  ______________________________________                                         Controls    35                 16                                               2 hours    56    69%↑   19   18↑                                  24 hours    40     5%↑   33  106↑                                  ______________________________________                                    

                  TABLE XLV                                                        ______________________________________                                         NK Cells                                                                       11:1                                                                                    #5              #6                                                    ______________________________________                                         Controls   18                7%                                                 2 hours   30    66%↑  7%                                                24 hours   17     5%↑  16%   129%↑                                 ______________________________________                                    

In view of the results of the human in vivo studies, it is again concluded and demonstrated that the enkephalins play a role as immunostimulatory agents; and when directly administrated to humans, act to immunomodulate and stimulate the immune system. It is further confirmed that this immunomoderation and stimulation is broadly directed to cell-mediated immunities including active T cell and NK cell populations, and as such the administration of the enkephalins and related composition according to the present invention have activity in regulating viruses, bacteria, fungi, tumerous cells and parasites and generally all immune deficiencies including deficiencies associated with aging. It is further felt that the enkephalins and endorphins, like interferons and interleukins, are the regulators (activators of other lymphokines) which act in concert in modulating and stimulating the immune system. It is proposed but not relied upon for purposes of this invention; therefore, the present invention is not limited thereby, that enkephalins activate T cells to release interleukin II which in turn activates the release of interferons and interleukin I and III, thus promoting a cascade of immunilogical effects.

Since Blalock et al, Proc. Natl. Acad. Sci. 77 10, 5972 (1980), reported antigenic and structural similarities among leukocyte interferon, ACTH, and gamma-endorphin, it can be implied that leukocyte interferon may be a precursor or is derived from a common precursor to these hormones. Furthermore, Laidow et al observed that both methionine-enkephalin and leucine-enkephalin as well as beta-endorphin inhibit phenylalanyl-t RNA synthetase, J.B.C. 255 4, 11908 (1980). The anti-tumor mechanisms of action of the enkephalins may be mediated through the immune systems by (1) T lymphocyte surface receptors, (2) interferon, interleukins, and other lymphokines. And more important, the same logic leads one to conclude that the observed significant protective effects of methionine-enkephalin and the anti-tumor activity of leucine-enkephalin of the present invention would be expected from their corresponding "precursor" molecules (e.g. beta-endorphin and dynorphin pro harmone) and molecules of similar structure derived from a common precursor. Further support for this statement can be found in and is implicit in the very presence of literature references indicating that "precursor" molecules bind to receptor sites of leukocytes (lymphocytes): Hazum et al, Science, 205, 7, 1033-1035 (1980). Thus for purposes of this invention the following closely related enkephalins and endorphins are to be considered equivalents:

                                      TABLE XLVI                                   __________________________________________________________________________     Peptide Structures                                                             __________________________________________________________________________     Met--enkephalin      Tyr--Gly--Gly--Phe--Met                                   Leu--enkephalin      Tyr--Gly--Gly--Phe--Leu                                   [Arg.sup.6 ]--Leu--enkephalin                                                                       Tyr--Gly--Gly--Phe--Leu--Arg                              [Arg.sup.6 ]--Met--enkephalin                                                                       Tyr--Gly--Gly--Phe--Met--Arg                              [Lys.sup.6 ]--Met--enkephalin                                                                       Tyr--Gly--Gly--Phe Met--Lys                               [Arg.sup.6 --Arg.sup.7 ]--Met--enkephalin                                                           Tyr--Gly--Gly--Phe--Met--Arg--Arg                         [Arg.sup.6 --Phe.sup.7 ]--Met--enkephalin                                                           Tyr--Gly--Gly--Phe--Met--Arg--Phe                         [Arg.sup.6 --Gly.sup.7 --Leu.sup.8 ]--Met--enkephalin                                               Tyr--Gly--Gly--Phe--Met Arg--Gly--Leu                     Alpha-Neo-endorphin  Tyr--Gly--Gly--Phe--Leu--Arg--Lys--Tyr--Pro--Lys          Beta-Neo-endorphin   Tyr--Gly--Gly--Phe--Leu--Arg--Lys--Tyr--Pro               Dynorphin            Tyr--Gly--Gly--Phe--Leu--Arg--Arg--Ile--Arg--Pro          Ph-8P (Dynorphin [1-8])                                                                             Tyr--Gly--Gly--Phe--Leu--Arg--Arg--Ile                    __________________________________________________________________________

The effective amount of endogenous peptide to be administered will vary depending on the circumstances and end result desired. Generally the dose ranges that can be used therapeutically are from about 0.001 to about 30 mg/kg. Preferably, dosage rates from about a quarter of a miligram of enkephalin per kilogram of body weight to as low as about 10⁻¹⁴ mg/kg are employed. The method of treatment and administration can be by any of the known routes including but not limited to: oral, i.v., i.m., s.c., aerosol, nasal, ophthalmic, or vaginal suppositories and the like. The enkephalins and endorphins can be used singly or in combination, or in combination with other known chemotherapeutic agents and/or antibiotics for the treatment of all forms of neoplastic activity, infections and/or related conditions.

Having thus described and exemplified the preferred embodiments with a certain degree of particularity, it is understood that the invention is not to be limited to the embodiments set forth herein for purposes of exemplification, but is to be limited only by the scope of the attached claims, including a full range of equivalents to which each element thereof is entitled. 

I claim:
 1. A process for stimulating the T-cell and NK cell immune activity comprising the step of administering in vivo an endogenous endorphin.
 2. A process of claim 1 wherein said endogenous endorphin is selected from the group consisting of beta-endorphin [61-91] and peptides of which beta-endorphin [61-91] is the precursor.
 3. A process of claim 1 wherein said endogenous endorphin is selected from the group consisting of the following: peptides; Tyr-Gly-Gly-Phe-Met; Tyr-Gly-Gly-Phe-Leu; Tyr-Gly-Gly-Phe-Leu-Arg; Tyr-Gly-Gly-Phe-Met-Arg; Tyr-Gly-Gly-Phe-Met-Lys; Tyr-Gly-Gly-Phe-Met-Arg-Arg; Tyr-Gly-Gly-Phe-Met-Arg-Phe; Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu; Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-Lys; Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro; Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro; Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile.
 4. A process of claim 1 wherein said endogenous endorphin is leucine-enkephalin.
 5. A process of claim 1 wherein said endogenous endorphin is methionine-enkephalin.
 6. A process of claim 4 or 5 wherein said enkephalin is administered in the range from about 0.001 to about 30 mg/kg.
 7. A process of claim 1 wherein endogenous endorphin is administered in a dosage range from about 0.25 mg/kg to about 10⁻¹⁴ mg/kg.
 8. A process of claim 2 wherein endogenous endorphin is administered in a dosage range from about 0.25 mg/kg to about 10⁻¹⁴ mg/kg.
 9. A process of claim 3 wherein endogenous endorphin is administered in a dosage range from about 0.25 mg/kg to about 10⁻¹⁴ mg/kg.
 10. A process of claim 4 wherein said leucine-enkephalin is administered in a dosage range from about 0.25 mg/kg to about 10⁻¹⁴ mg/kg.
 11. A process of claim 4 wherein said methionine-enkephalin is administered in a dosage range from about 0.25 mg/kg to about 10⁻¹⁴ mg/kg. 